Our overall goal is to determine the mechanisms by which CD8+ cytotoxic T lymphocyte (CTL) and CD4+ T helper responses can be elicited against highly conserved internal virion proteins of HIV-1 Gag. Toward this goal, a novel development is the construction of DNA expression vectors, which enable us to express Gag in a Rev and RRE independent and species independent fashion. Using this new method, we will systematically evaluate the expression of, and immune response to, different subcellularly targeted forms of HIV-1 Gag to maximize the induction of CTL and T helper responses against Gag in mice. We will carry out two specific aims: (1). Characterization of the ability of various HIV-1 Gag expression vectors to induce CD8+ CTL. HIV-1 Gag expression vectors which enable us to express HIV-1 Gag molecules in mouse cells in the absence of Rev and RRE have been generated. Derivative Gag expression vectors, which are targeted to different intracellular and extracellular compartments for antigen processing and presentation will be constructed and evaluated. These different forms of Gag include: (a) wild type Gag that assembles into particles which are released from cells efficiently; (b) mutant Gags that fail to target to plasma membrane and remain in the cytoplasm; (c) mutant Gags that fail to assemble into particles; (d) mutant Gags that are rapidly degraded intracellularly, (e) secreted but non-particle forms of Gag; (f) cell surface anchored forms of Gag. (A) Various forms of HIV-11 Gag molecules will be expressed in p815 cells and compared as CTL target cells. (B) DNA vectors expressing various forms of HIV-1 Gag molecules will be tested for CTL induction in mice. (2). Characterization of the ability of various HIV-1 Gag expression vectors that will optimize T helper response especially those that could enhance CTL response. (A) The different forms of HIVA Gag DNA vaccine described above will also be evaluated for induction of T helper responses after DNA immunization in mice by measuring T cell proliferative and cytokine responses after Gag stimulation in vitro. (B) A CTLA-4-HIV-1 Gag fusion molecule encoded by a single DNA vaccine vector will be evaluated. This strategy will allow specific targeting of Gag molecules to antigen presentation cells for MHC class 11 presentation. (C) Combination studies by using DNA vectors identified from this specific aim with DNA vectors identified from specific aim 1.